Delayed tooth movement in Runx2+/- mice associated with mTORC2 in stretch-induced bone formation.

Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and is activated by mechanical stress to promote osteoblast function. Cleidocranial dysplasia (CCD) is caused by mutations of RUNX2, and CCD patients exhibit malocclusion and often need orthodontic treatment. However, treatment is difficult because of impaired tooth movement, the reason of which has not been clarified. We examined the amount of experimental tooth movement in Runx2+/- mice, the animal model of CCD, and investigated bone formation on the tension side of experimental tooth movement in vivo. Continuous stretch was conducted to bone marrow stromal cells (BMSCs) as an in vitro model of the tension side of tooth movement. Compared to wild-type littermates the Runx2+/- mice exhibited delayed experimental tooth movement, and osteoid formation and osteocalcin (OSC) mRNA expression were impaired in osteoblasts on the tension side of tooth movement. Runx2 heterozygous deficiency delayed stretch-induced increase of DNA content in BMSCs, and also delayed and reduced stretch-induced alkaline phosphatase (ALP) activity, OSC mRNA expression, and calcium content of BMSCs in osteogenic medium. Furthermore Runx2+/- mice exhibited delayed and suppressed expression of mammalian target of rapamycin (mTOR) and rapamycin-insensitive companion of mTOR (Rictor), essential factors of mTORC2, which is regulated by Runx2 to phosphorylate Akt to regulate cell proliferation and differentiation, in osteoblasts on the tension side of tooth movement in vivo and in vitro. Loss of half Runx2 gene dosage inhibited stretch-induced PI3K dependent mTORC2/Akt activity to promote BMSCs proliferation. Furthermore, Runx2+/- BMSCs in osteogenic medium exhibited delayed and suppressed stretch-induced expression of mTOR and Rictor. mTORC2 regulated stretch-elevated Runx2 and ALP mRNA expression in BMSCs in osteogenic medium. We conclude that Runx2+/- mice present a useful model of CCD patients for elucidation of the molecular mechanisms in bone remodeling during tooth movement, and that Runx2 plays a role in stretch-induced proliferation and osteogenesis in BMSCs via mTORC2 activation.

Novel strategies for expansion of tooth epithelial stem cells and ameloblast generation.

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs). Humans cannot repair or regenerate enamel, due to early loss of tooth epithelial SCs. Contrarily in the mouse incisors, epithelial SCs are maintained throughout life and endlessly generate ameloblasts, and thus enamel. Here we isolated Sox2-GFP+ tooth epithelial SCs which generated highly cellular spheres following a novel in vitro strategy. This system enabled analysis of SC regulation by various signaling molecules, and supported the stimulatory and inhibitory roles of Shh and Bmp, respectively; providing better insight into the heterogeneity of the SCs. Further, we generated a novel mouse reporter, Enamelin-tdTomato for identification of ameloblasts in live tissues and cells, and used it to demonstrate presence of ameloblasts in the new 3D co-culture system of dental SCs. Collectively, our results provide means of generating 3D tooth epithelium from adult SCs which can be utilized toward future generation of enamel.

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

Mesenchymal Wnt/ß-catenin signaling limits tooth number.

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/ß-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/ß-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/ß-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/ß-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/ß-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2, a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2/Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/ß-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations.

Early epithelial signaling center governs tooth budding morphogenesis.

During organogenesis, cell fate specification and patterning are regulated by signaling centers, specialized clusters of morphogen-expressing cells. In many organs, initiation of development is marked by bud formation, but the cellular mechanisms involved are ill defined. Here, we use the mouse incisor tooth as a model to study budding morphogenesis. We show that a group of nonproliferative epithelial cells emerges in the early tooth primordium and identify these cells as a signaling center. Confocal live imaging of tissue explants revealed that although these cells reorganize dynamically, they do not reenter the cell cycle or contribute to the growing tooth bud. Instead, budding is driven by proliferation of the neighboring cells. We demonstrate that the activity of the ectodysplasin/Edar/nuclear factor κB pathway is restricted to the signaling center, and its inactivation leads to fewer quiescent cells and a smaller bud. These data functionally link the signaling center size to organ size and imply that the early signaling center is a prerequisite for budding morphogenesis.

Effect of Estrogen and Food Hardness on Metabolism and Turnover of Condylar Cartilage.

AIMS: To clarify the effect of estrogen and food hardness on condylar cartilage and the cartilage-bone interface. METHODS: A total of 56 rats were divided into four groups: (1) ovariectomized rats fed a normal (pressed pellet) food, (2) ovariectomized rats fed a soft (powder) food, (3) control rats fed a normal (pressed pellet) food, and (4) control rats fed a soft (powder) food. Some rats (n = 29) were sacrificed at the age of 67 days and others (n = 27) at the age of 87 days, and then 5-µm-thick sagittal paraffin sections were prepared from each temporomandibular joint (TMJ). Toluidine blue staining, in situ hybridization with type X collagen, terminal deoxynucleotidyl transferase and deoxyuridine triphosphate nick end labeling (TUNEL-assay), and tartrate-resistant acid phosphatase (TRAP) histochemistry were performed. Immunohistochemical analyses included cathepsin K, adiponectin, proliferating cell nuclear antigen (PCNA), and type X collagen staining. Analysis of variance and appropriate post-hoc tests were used in all analyses. RESULTS: Ovariectomy and normal food consistency increased the thickness of condylar cartilage (P < .001), PCNA expression (P < .001) and type X collagen expression (P < .001). Ovariectomy decreased the number (P < .05) and size of osteoclasts (P < .01). Soft food increased the number of cartilage cells stained positively against adiponectin (P < .01). CONCLUSION: Decreased estrogen level and normal food hardness increase the thickness of condylar cartilage by various mechanisms.

Mesenchymal Wnt/ß-Catenin Signaling Controls Epithelial Stem Cell Homeostasis in Teeth by Inhibiting the Antiapoptotic Effect of Fgf10.

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/ß-catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/ß-catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no ß-catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO-treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/ß-catenin. Stem Cells 2015;33:1670-1681.

Glial origin of mesenchymal stem cells in a tooth model system.

Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair. The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium. It has been thought for decades that the dental mesenchymal stem cells giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue. Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.

Expression of the stem cell marker, SOX2, in ameloblastoma and dental epithelium.

Ameloblastomas are locally invasive odontogenic tumors that exhibit a high rate of recurrence and often associate with the third molars. They are suggested to originate from dental epithelium because the tumor cells resemble epithelial cells of developing teeth. Expression of the transcription factor SOX2 has been previously localized in epithelial stem and progenitor cells in developing teeth as well as in various tumors. Here, we show that SOX2 is expressed in the epithelial cells of follicular and plexiform ameloblastomas. SOX2 was localized in the dental lamina of developing human primary molars. It was also expressed in the fragmented dental lamina associated with the third molars and in the epithelium budding from its posterior aspect in mice. However, no SOX2 expression was detected in either Hertwig's epithelial root sheath directing the formation of roots or in the epithelial cell rests of Malassez covering the completed roots. SOX2 was associated with supernumerary tooth formation in odontoma-like tumors induced by Wnt signal activation in mice. We propose that SOX2 functions in maintaining the progenitor state of epithelium in ameloblastomas and that ameloblastomas may originate from SOX2-expressing dental lamina epithelium.

Sox2 marks epithelial competence to generate teeth in mammals and reptiles.

Tooth renewal is initiated from epithelium associated with existing teeth. The development of new teeth requires dental epithelial cells that have competence for tooth formation, but specific marker genes for these cells have not been identified. Here, we analyzed expression patterns of the transcription factor Sox2 in two different modes of successional tooth formation: tooth replacement and serial addition of primary teeth. We observed specific Sox2 expression in the dental lamina that gives rise to successional teeth in mammals with one round of tooth replacement as well as in reptiles with continuous tooth replacement. Sox2 was also expressed in the dental lamina during serial addition of mammalian molars, and genetic lineage tracing indicated that Sox2(+) cells of the first molar give rise to the epithelial cell lineages of the second and third molars. Moreover, conditional deletion of Sox2 resulted in hyperplastic epithelium in the forming posterior molars. Our results indicate that the Sox2(+) dental epithelium has competence for successional tooth formation and that Sox2 regulates the progenitor state of dental epithelial cells. The findings imply that the function of Sox2 has been conserved during evolution and that tooth replacement and serial addition of primary teeth represent variations of the same developmental process. The expression patterns of Sox2 support the hypothesis that dormant capacity for continuous tooth renewal exists in mammals.

Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes.

BACKGROUND: Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. RESULTS: Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. CONCLUSIONS: Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development.

Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ progenitors.

The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal.

Signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages.

Teeth develop as ectodermal appendages from epithelial and mesenchymal tissues. Tooth organogenesis is regulated by an intricate network of cell-cell signaling during all steps of development. The dental hard tissues, dentin, enamel, and cementum, are formed by unique cell types whose differentiation is intimately linked with morphogenesis. During evolution the capacity for tooth replacement has been reduced in mammals, whereas teeth have acquired more complex shapes. Mammalian teeth contain stem cells but they may not provide a source for bioengineering of human teeth. Therefore it is likely that nondental cells will have to be reprogrammed for the purpose of clinical tooth regeneration. Obviously this will require understanding of the mechanisms of normal development. The signaling networks mediating the epithelial-mesenchymal interactions during morphogenesis are well characterized but the molecular signatures of the odontogenic tissues remain to be uncovered.

Ectodysplasin and Wnt pathways are required for salivary gland branching morphogenesis.

The developing submandibular salivary gland (SMG) is a well-studied model for tissue interactions and branching morphogenesis. Its development shares similar features with other ectodermal appendages such as hair and tooth. The ectodysplasin (Eda) pathway is essential for the formation and function of several ectodermal organs. Mutations in the signaling components of the Eda pathway lead to a human syndrome known as hypohidrotic ectodermal dysplasia (HED), which is characterized by missing and malformed teeth, sparse hair and reduced sweating. Individuals with HED suffer also from dry mouth because of reduced saliva flow. In order to understand the underlying mechanism, we analyzed salivary gland development in mouse models with altered Eda pathway activities. We have found that Eda regulates growth and branching of the SMG via transcription factor NF-κB in the epithelium, and that the hedgehog pathway is an important mediator of Eda/NF-κB. We also sought to determine whether a similar reciprocal interplay between the Eda and Wnt/ß-catenin pathways, which are known to operate in other skin appendages, functions in developing SMG. Surprisingly and unlike in developing hair follicles and teeth, canonical Wnt signaling activity did not colocalize with Edar/NF-κB in salivary gland epithelium. Instead, we observed high mesenchymal Wnt activity and show that ablation of mesenchymal Wnt signaling either in vitro or in vivo compromised branching morphogenesis. We also provide evidence suggesting that the effects of mesenchymal Wnt/ß-catenin signaling are mediated, at least in part, through regulation of Eda expression.

Cooperation of nectin-1 and nectin-3 is required for normal ameloblast function and crown shape development in mouse teeth.

Nectins are immunoglobulin-like cell adhesion proteins and their interactions recruit various cell-cell junctions. Mutations in human NECTIN-1 cause an ectodermal dysplasia syndrome, but Nectin-1 null mice have only slight defects in teeth, suggesting compensation by other nectin(s). We observed overlapping expression of nectin-3 with nectin-1 and enamel abnormality in the nectin-3 mutant. We, therefore, generated nectin-1;nectin-3 compound mutants. However, all teeth developed and no significant dental abnormalities were observed before birth. At postnatal day 10, the upper molars of compound mutants exhibited conical crown shape and retarded enamel maturation. Nectin-1 was expressed in ameloblasts whereas nectin-3 was expressed in neighboring stratum intermedium cells at this stage. The immunohistochemical localization and electron microscopical observations indicated that the desmosomal junctions between stratum intermedium and ameloblasts were significantly reduced. These results suggest that heterophilic interaction between nectin-1 and nectin-3 recruits desmosomal junctions, and that these are required for proper enamel formation.

Wnt/ß-catenin signaling in the dental mesenchyme regulates incisor development by regulating Bmp4.

Loss- and gain-of function approaches modulating canonical Wnt/ß-catenin activity have established a role for the Wnt/ß-catenin pathway during tooth development. Here we show that Wnt/ß-catenin signaling is required in the dental mesenchyme for normal incisor development, as locally restricted genetic inactivation of ß-catenin results in a splitting of the incisor placode, giving rise to two incisors. Molecularly this is first associated with down-regulation of Bmp4 and subsequent splitting of the Shh domain at a subsequent stage. The latter phenotype can be mimicked by ectopic application of the BMP antagonist Noggin. Conditional genetic inactivation of Bmp4 in the mesenchyme reveals that mesenchymal BMP4 activity is required for maintenance of Shh expression in the dental ectoderm. Taken together our results indicate that ß-catenin together with Lef1 and Tcf1 are required to activate Bmp4 expression in order to maintain Shh expression in the dental ectoderm. This provides a mechanism whereby the number of incisors arising from one placode can be varied through local alterations of a mesenchymal signaling circuit involving ß-catenin, Lef1, Tcf1 and Bmp4.

Notch signalling is required for the survival of epithelial stem cells in the continuously growing mouse incisor.

The Notch pathway regulates the renewal and fate decisions of stem cells in multiple tissues. Notch1, -2, as well as the Notch target gene Hes1 are expressed in the putative stem cells in the continuously growing mouse incisors, but so far there has not been any evidence for a function of the Notch pathway in the regulation of the incisor stem cells. We have analysed the effects of the Notch pathway inhibitor DAPT on the maintenance, proliferation, and differentiation of the epithelial stem cells in explant cultures of the mouse incisor. The proximal part of the incisor containing the cervical loop stem cell niche was dissected from newborn mice and cultured for 2-6 days in vitro. DAPT inhibited the expression of Notch target gene Hes1 in the cervical loop indicating that Notch signalling was inhibited in the putative stem cells. The most striking effect of DAPT was a significant reduction in the size of the cervical loop. DAPT caused a marked but partially reversible decrease in cell proliferation, as well as massive apoptosis in the epithelial stem cell niche. Interestingly, restricted apoptosis was detected within the Notch expressing putative stem cells also in the control cultures as well as in incisors in vivo, suggesting that apoptosis may be a mechanism regulating the size of the epithelial stem cell pool in the incisor. The differentiation of the epithelial cells into enamel-forming ameloblasts was not affected by DAPT but the number of preameloblasts was progressively decreased during culture period reflecting the depletion of stem and progenitor cells. Our results indicate that Notch signalling is required for epithelial stem cell survival and enamel formation in the continuously growing mouse incisor.

Patterns of Wnt pathway activity in the mouse incisor indicate absence of Wnt/beta-catenin signaling in the epithelial stem cells.

The Wnt pathway is crucial for tooth development as shown by dental defects caused by impaired Wnt signaling in mouse and human. We investigated Wnt signaling in continuously growing mouse incisors focusing on epithelial stem cells. Ten Wnt ligands were expressed both in the dental epithelium and mesenchyme, and were associated mainly with odontoblast and ameloblast differentiation. Wnt/beta-catenin activity was detected in mesenchyme in BATgal and TOPgal reporter mice while Axin2, also a reporter of Wnt/beta-catenin signaling, was expressed additionally in the epithelium. Axin2 was, however, excluded from the epithelial stem cells in the cervical loop. Interestingly, these cells expressed specifically Lgr5, a Wnt target gene and stem cell marker in the intestine, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt signaling in the incisor. We conclude that epithelial stem cells in the mouse incisors are not regulated directly by Wnt/beta-catenin signaling.

Runx1 is involved in the fusion of the primary and the secondary palatal shelves.

Runx1 is expressed in medial edge epithelial (MEE) cells of the palatal shelf. Conditionally rescued Runx1(-/-) mice showed limited clefting in the anterior junction between the primary and the secondary palatal shelves, but not in the junction between the secondary palates. In wild type mice, the fusing epithelial surface exhibited a rounded cobblestone-like appearance, while such cellular prominence was less evident in the Runx1 mutants. We also found that Fgf18 was expressed in the mesenchyme underlying the MEE and that locally applied FGF18 induced ectopic Runx1 expression in the epithelium of the palatal explants, indicating that Runx1 was induced by mesenchymal Fgf18 signaling. On the other hand, unpaired palatal explant cultures revealed the presence of anterior-posterior (A-P) differences in the MEE fates and fusion mechanism. Interestingly, the location of anterior clefting in Runx1 mutants corresponded to the region with different MEE behavior. These data showed a novel function of Runx1 in morphological changes in the MEE cells in palatal fusion, which is, at least in part, regulated by the mesenchymal Fgf signaling via an epithelial-mesenchymal interaction.